Cytotoxic protein and utilization thereof

ABSTRACT

This invention relates to a new cytotoxic protein (M toxin, mucous layer devastating toxin) produced by  Helicobacter pylori  and the use. 
     This invention provides a cytotoxic protein (M toxin) produced by  Helicobacter pylori , a partial peptide, and an antitumor agent containing the cytotoxic protein. The protein is obtained by culturing a transformant which is transformed with a recombination vector containing DNA coding the cytotoxin protein. This invention provides further the use of the protein.

FIELD OF THE INVENTION

This invention relates to a new cytotoxic protein (M toxin, mucous layerdevastating toxin) produced by Helicobacter pylori and the use.

BACKGROUND ART

It has been considered that the human gets many gastritis, gastric ulcerand gastric cancer by Helicobacter pylori. A distinct direct cytotoxicfactor causing destruction of gastric epithelial cells and irreversiblecell death at the beginning of these diseases has not been specified. Afactor changing pH environment and immunity reaction in the stomach, afactor adhering to gastric epithelial cells by Helicobacter pylori, ormove properties of bacteria themselves has been indicated as a factordeveloping such diseases. However, it has been unclear hitherto thatgastric mucosal destruction triggering gastritis, gastric ulcer andgastric cancer is injured in any process, or what is a directresponsible factor to the gastric mucosal destruction. The only is thatvacuolating toxin having cytotoxicity is isolated, but it has weekcytotoxic activity and reversible cytotoxic activity. As a fatalcytotoxic factor of a pathogenic factor, it has not been found in vivoand in vitro.

Many researchers estimate that Helicobacter pylori, as described in theabove, in the environment stomach in vivo, secretes a direct cytotoxicfactor for gastric mucosal cells. Considering the importance ofdiseases, all sequences of the gene is confirmed in 1996. However, inspite of the use of serum, it is impossible to isolate and identify thepresumable toxin due to separation conditions, with difficult cultureconditions and purification conditions and unfixed valuation systems ofthe toxin.

Problems to be Solved by the Invention

One problem is that responsible protein causing gastritis, gastric ulcerand gastric cancer by Helicobacter pylori infection is found, a massproduction method of the toxic protein is established, new M toxin isidentified, and diagnosis and screening method is established. Usingthese methods, it is desired that responsible toxin of a cytotoxicfactor for gastric mucosal cells is controlled, and preventive andtreating agents of gastritis, gastric ulcer, gastric cancer and the likeare developed and a method for using the toxin is found.

DISCLOSURE OF THE INVENTION

Inventors of this invention have studied earnestly to resolve theabove-mentioned problem, and identified a new toxin that causesirreversible cell death when Helicobacter pyloriis cultured underserum-free conditions that is similar to environment in the stomachdifferent from usual. It has been found that this toxin has a toxicpower 1000 to 100000 times per unit of above-mentioned vacuolatingtoxin, and it causes irreversible cell death for various warm-bloodedanimal cells comprising not only gastric epithelial cells but alsoimmunocytes. The inventors of this invention have repeatedly researchedon these views to attain to the present invention.

Namely, this invention provides the following. (1) A cytotoxic proteincomprising a protein having at least 70% or more identity for the aminoacid sequence represented by SEQ ID No. 1. (2) A partial peptide of thecytotoxic protein described in claim 1, characterized in that theprotein has the same cytotoxic activity as that of the amino acidsequence represented by SEQ ID No. 1. (3) The cytotoxic protein of claim1 or claim 2 wherein the protein is produced with Helicobacter pylori.(4) The cytotoxin protein of claim 1 or claim 2 wherein the protein isobtained by culturing a transformant which is transformed with arecombination vector containing DNA of SEQ ID No. 2 coding the cytotoxinprotein of claim 1 or 2. (5) The cytotoxin protein of claim 4 whereinthe transformant is deposited by deposition No. FERM BP-8218 at NationalInstitute of Advanced Industrial Scienc and Technology (IPOD). (6) Anantitumor agent which contains the cytotoxic protein of claim 1 or 2.(7) A monoclonal antibody specific against the cytotoxic protein whichis obtained by immunization of the cytotoxic protein of claim 1 or 2against a mammal. (8) The monoclonal antibody of claim 7 which isproduced with a hybridoma done of deposition No. FERM BP-8222. (9) Themonoclonal antibody of claim 7 which is produced with a hybridoma cloneof deposition No. FERM BP-8223. (10) The monoclonal antibody of claim 7which is produced with a hybridoma done of deposition No. FERM BP-8224.(11) A polyclonal antibody specific against the cytotoxic protein whichis obtained by immunization of the cytotoxic protein of claim 1 or 2against a mammal. (12) A method for detecting and diagnosing thecytotoxic protein of claim 1 or 2 wherein the monoclonal antibody orpolyclonal antibody described in any one of claims 7-11 is used. (13) Anagent for preventing and treating gastric cancer, gastritis and gastriculcer triggered by the cytotoxic protein of claim 1 or 2, wherein themonoclonal antibody or polyclonal antibody described in any one ofclaims 7-11 are used. (14) A method for screening a compound promotingor inhibiting activity of the protein of claim 1 or 2, wherein cellmultiplication inhibition activity, cytotoxic activity or cell death isjudged by comparison between negative or positive control groups with awarm-blooded animal cell. (15) A kit for screening a compound or itssalt promoting or inhibiting activity of the protein of claim 1 or 2,wherein the protein of claim 1 or 2 is comprised. (16) A compound or itssalt promoting or inhibiting activity of the protein of claim 1 or 2,wherein the compound is obtained by the screening method of claim 14 orwith the kit for screening of claim 15. (17) A medicine containing acompound or its salt, wherein the compound has activity inhibitingcytotoxic activity of warm-blooded animal cells with the protein ofclaim 1 or 2. (18) A medicine of claim 17, which is an agent forpreventing or treating gastritis, gastric ulcer, gastric cancer and adisease showing that it is caused with M toxin by the screening methodof cliam 14 or with the screening kit of claim 15.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an anion exchange chromatography of the sample obtained inExample 1. a shows activity of M toxin for each fraction and absorbancesof the elute protein. b shows SDS-PAGE by silver stain of fraction 16 tofraction 22 in the chromatography of a. c shows bands of protein toxintransferred to a PVDF membrane by electroblotting. d and e showmorphologic changes of HeLa cell 24 hours after exposure to the controlextract and the cytotoxin including extract at a concentration of 1 nM,respectively. Scale bar, 50 μm.

FIG. 2 shows cell morphologic change with the gene recombinant toxinobtained in Example 3. a shows a negative contlol of HeLa cells after 6hours. b and c show HeLa cells after 3 hours and after 6 hours of 5 nMaddition of recombinant M toxin. d shows CRL7407(ATCC) normal humangastric cells of negative control after 6 hours. e and f show CRL7407cells after 3 hours and 6 hours of 5 nM addition of recombinant M toxin.

FIG. 3 shows sensitivity for M toxin of several kinds of cancer cells inExample 3. It was determined by a WST method. X axis shows concentrationof M toxin in a substrate, and Y axis shows directly absorbance ofwavelength 415 nm or by a relative ratio when the absorbance of anegative control is estimated as 100%. HLF: rat hepatoma. colon 26:carcinoma of the mouse colon.

FIG. 4 is as shown in FIG. 3. T24: human cancer of the bladder. OVK18:human ovarian cancer. KLM-1: human pancreatic cancer. A-549: human lungcancer. Ca9-22: human cancer of the gum. CRL1500: human cancer of thebreast.

FIG. 5 shows western blotting of monoclonal antibody which is conductedin Example 8. All dilution magnifications of cultured supernatants ofhybridoma cells are 20 times.

FIG. 6 shows M toxin activity with calcium alginate as an adsorbent inExample 9. The negative control contains 10 mM Tris buffer pH7.7 aloneas a substrate, and the positive control contains a substrate and 10 mMM toxin. X axis shows each fraction numaber passed through a calciumalginate column. An average concentration of each fraction is 10 nM. andany one of the fractions shows at least 10 nM or more. When redcorpuscles are destroyed, hemoglobin concentration in the solution isincreased. Y axis shows 415 nm absorbance.

FIG. 7 shows, in Example 10, % viability of HeLa cells showing M toxininhibition activity by adsorption of activated carbon in Example 10. Xaxis shows each elute fraction number of column. M toxin concentrationsare average 10 nM and any one of them is at least 10 nM or more. Y axisshows % obtained by dividing the value of the toxicity of each fractionby a positive control which is measured by a WST method.

FIG. 8 shows as shown in FIG. 7. It shows % viability in HeLa cells ofCM-cellulose and calcium alginate showing M toxin inhibition activity byadsorption in Example 10.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein having the same amino acid sequence or substantiallyidentical amino acid sequence with the amino acid sequence representedby SEQ ID No.1 of this invention may be a protein originating frombacteria strains of Helicobacter pylori, for example, NCTC 11637, NCTC11916, DT 61A, NCTC 11639, R85-13 6P, R85-13-12F, R85-13-11P,T81213-NTB, J99, 4, U2-185D08, MC903, MC123, Tx30a, 26695, UA 1182 andthe like, or my be a synthesized protein.

As substantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID No. 1, amino acid sequences having homology ofabout 70% or more, preferably about 80% or more, more preferably about90% or more, further more preferably about 95% or more can beexemplified. As a protein having substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID No.1, aprotein having substantially the same amino acid sequence andsubstantially the same activity as that of the protein having the aminoacid sequence represented by SEQ ID No. 1 can be exemplified. Assubstantially the same activity, a cell multiplication inhibitionactivity, a cytotoxic activity or an activity causing cell death can beexemplified. The term of substantially the same means that theseactivities have tha same quality, for example, in physiologicalchemistry or pharmacology. Accordingly, it is preferred that theactivity of cytotoxin and the like has the same quality, for example,about 0.1-100 times, preferably about 0.5-10 times, more preferablyabout 0.5-2 times. The degree of these activities or quantity factors ofmolecular weights of proteins may be different. The activities of a cellmultiplication inhibition activity, a cytotoxic activity or an activitycausing cell death can be determined by well-known methods, for example,by the screening method undermentioned.

As the protein of this invention, the following proteins can beexemplified: an amino acid sequence deleting 1-150 (preferably 1-50)amino acids in the amino acid sequence represented by SEQ ID No.1; anamino acid sequence adding 1-100 (preferably 1-30) amino acids in theamino acid sequence represented by SEQ ID No. 1; an amino acid sequenceinserting 1-50 (preferably 1-30) amino acids in the amino acid sequencerepresented by SEQ ID No. 1; an amino acid sequence that 1-50(preferably 1-30) amino acids in the amino acid sequence represented bySEQ ID No. 1 are substituted by the other amino acids; or a proteincontaining a combination of these amino acid sequences, what is calledmucin.

When an amino acid sequence is iserted, deleted or substituted asdescribed in the above, the position of insertion, deletion orsubstitution is not specifically limited.

In the protein of this specification, according to a usual practice, theleft side is an N end (an amino end) and the right side is a C end (acarboxyl end). In the proteins of this invention, which comprises theprotein having the amino acid sequence represented by SEQ ID No.1, the Cend is usually a carboxyl group (—COOH) or a carboxylate (—COO—), but itmay be an amid (—CONH₂) or an ester group (—COOR), wherein R is a C₁₋₆alkyl group of methyl, ethyl, n-propyl, isopropyl, n-butyl and the like,a C₃₋₈ cycloalkyl group of cyclopentyl, cyclohexyl and the like, a C₆₋₁₂aryl group of phenyl, α-naphthyl and the like, benzyl, phenetyl and thelike, a phenyl-C₁₋₂ alkyl group of benzyl, phenetyl and the like, or aC₇₋₁₄ aralkyl group of an α-naphthyl-C₁₋₂ alkyl group ofα-naphthylmethyl and the like. When the protein of this invention has acarboxyl group (or carboxylate) at a position other than the C end,proteins having an amidic or esterified group are contained in theprotein of this invention. As the ester, the ester of theabove-mentioned C end may be used. In the proteins of this invention,further, there is a protein that the amino group of an amino acidresidue (for example, a methionin residue) at the N end is protected bya protective group (for example, a C₁₋₆ acyl group of a C₁₋₆ alkanoylgroup or the like of a formyl group, acetyl group or the like); aprotein that the glutamic acid residue of the N end produced by incisionin vivo is changed into pyrroglutamic acid group; a protein that thesubstituent group on the side chain of an amino acid in the molecular(for example, —OH, —SH, an amino group, an imidazole group, an indolgroup, a guanidino group or the like) is protected by a suitableprotective group (for example, a C₁₋₆ acyl group or the like of C₁₋₆alkanoyl group or the like of a formyl group); or a conjugated proteinor the like of so-called glycoproteins that the sugar chain is bound.

As the partial peptide of the protein of this invention, it may be apartial peptide of the above-mentioend proteins of this invention, andpreferably, it has similar activities (for example, a cellmultiplication inhibition activity, a cytotoxic activity or the like) tothe proteins of this invention. As an example, a peptide of an aminoacid sequence having at least 20% or more, preferably 50% or more, morepreferably 70% or more, further preferably 90% or more, and mostpreferably 95% or more in the amino acid sequence of this invention, andhaving a cell multiplication inhibition activity, a cytotoxic activityor an activity causing cell death can be utilized. The partial peptideof this invention may be the following peptides: 1-5 (prederably 1-3)amino acids are deleted in the amino acid sequence; 1-10 (preferably 1-5(more preferably 1-3)) amino acids added to the amino acid sequence; 1-5(preferably 1-3) amino acids are inserted into the amino acid sequence;or 1-5 (preferably 1-3) amino acids are substituted by the other aminoacids.

Although the partial peptide of this invention has usually a carboxylgroup (—COOH) or carboxylate (—COO—) at the C end, as shown in the aboveproteins of this invention, the C end may be amide (—CONH₂) or ester(COOR) wherein R is the same meaning as shown in the above. In thepartial peptide of this invention, further, as shown in the aboveproteins of this invention, there is a peptide that the amino group ofan amino acid residue (for example, a methionin residue) at the N end isprotected by a protective group; a protein that the glutamic acidresidue of the N end produced by incision in vivo is changed intopyrroglutamic acid group; a protein that the substituent group on theside chain of an amino acid in the molecular is protected by a suitableprotective group; or a conjugated protein or the like of so-calledglycoproteins that the sugar chain is bound. The partial peptide of thisinvention can be used as an antigen for constructing an antibody, sothat a cell multiplication inhibition activity, a cytotoxic activity orthe like is no necessarily required.

As the salt of the protein or partial peptide of this invention, saltsof physiologically allowable acids (for example, inorganic acids ororganic acids) or bases (for example, alkali metal salts) can be used,and preferably physiologically allowable acid addition salts, can beused. As such salts, for example, there are salts of inorganic acids(for example, hydrochloric acid, phosphoric acid, hydrobromic acid,sulfuric acid), organic acids (for example, acetic acid, formic acid,propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid,citric acid, malic acid, oxalic acid, benzoic acid, methane sulfonicacid, benzene sulfonic acid). The proteins and the salts of thisinvention can be produced by well-known preparation methods of proteinsfrom any kinds of strains of above-mentioned Helicobacter pylori, or byculturing a transformant containing DNA that after-mentioned protein iscoded. They further can be produced in accordance with after-mentionedmethod for syntheizing peptides. When the proteins are produced from anykinds of strains of Helicobacter pylori, proteins in bacterial cells arecentrifuged off by ultrsonic crushing, then they are extracted byammonium sulfate precipitation or the like, and the extracted liquid ispurified and separated by combined chromatography such as ion exchangechromatography and hydrophobic chromatography.

In the synthesis of the protein, the partial peptide, salts thereof oramide thereof of this invention, commertially available resins forsynthesizing proteins can be used. As such resins, the following resinscan be exemplified: chloromethyl resin, hydroxymethyl resin,benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzylalcoholresin, 4-methylbenzhydryl-amine resin, PAM resin,4-hydroxymethylmethylphenylacetoamidemethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin. Using suchresins, an amino acid that α-amino groups and functional groups of sidechains are suitably protected is condensed on the resin to meet to thedesired protein sequence by a well-known condensation method. At the endof reaction, the protein is cut off from the resin and severalprotecting groups are deleted. Then, the desired proteins or amidesthereof are obtained by a method for forming internal disulfide bonds ina high dilution solution. Concerning the above-mentioned condensation ofprotected amino acids, several kinds of activated agents for proteinsynthesis can be used, and particularly, carbodiimides can be used. Assuch carbodiimides, DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and the like can be used.In activation by these carbodiimides, the protected amino acid can bedirectly added to the resin with an additional agent for controllingracemization (for example, HOBt or HOOBt), or it can be added to theresin after previous activation of the protected amino acid as asymmetric acid anhydride or, HOBt ester or HOOBt ester.

As the solvent used in the activation of the protected amino acid or thecondensation with the resin, it can be selected from known solventsusable for the protein condensation reaction. As such solvent, acidamides such as N,N-dimethylformamide, N,N-dimethylacetoamide andN-methylpyrolidone, halogenated hydrocarbons such as methylene chlorideand chloroform, alcohols such as trifuloroethanol, sulfoxydes such asdimethylsulfoxide, pyridine, eters such as dioxane and tetrahydrofuran,nitriles such as acetonitrile and propionitrile, esters such as methylacetate and ethyl acetate, and mixtures thereof can be used. Thereaction temperature is suitably selected from the known ranges that canbe used in the reaction for forming protein bonds, and usually, it issuitably selected from −20° C.˜50° C. Activeted amino acid derivativesare usually used by 1.5-4 times of the mole equivalent. As a test resultof ninhydrin reaction, when the condensation is insufficient, thecondensation reactions can be repeated without the deletion of theprotected groups to conduct sufficient condensation. When sufficientcondensation can not be conducted by the repeated reactions, unreactedamino acids are acetylated with acetic anhydride or acety imidazole, sothat subsequent reactions have no influence.

As the protected groups of amino groups of starting materials, forexample, Z, Boc, t-pentyloxycarhonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, Fmoc and the like can be used. The carboxylgroups can be protected, for example, by alkyl-esterification (forexample, alkylesteration of straight, branched or cyclic chains ofmethyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl and 2-adamantyl), aralkylesteration (forexample, benzylester, 4-nitrobenzylester, 4-methoxybenzylester,4-chlorobenzylester, benzhydrylester), phenacylesteration,benzyloxycarbonylhydradidation, t-butoxycarbonylhydradidation,tritylhydradidation and the like. The hydroxyl group of serine can beprtected, for example, by esterification or etheration. As the suitablegroups of this esterification, for example, lower (C₁₋₆) alkanoyl groupssuch as an acetyl group, an acyl group such as a benzoyl group, orgroups derived from carbonate such as a benzyloxycarbonyl group andethoxycarbonyl group can be used. As groups suitable to the etheration,a benzyl group, a tetrahydropyranyl group and a t-butyl group can beexemplified. As the protected groups of phenolic hydroxyl groups oftyrosine, for examle, Bzl, C₁₂-Bzl, 2-nitrobenzyl, Br-Z and t-butyl canbe used. As the protected groups of imidazole of histidine, for example,Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl,Bum, Boc, Trt and Fmoc can be used.

As the activated carboxyl groups of starting materials, for example,corresponding acid anhydrides, azides and activated esters (esters ofalcohol (for example, pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethylalcohol, paranitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide and HOBt)) can be used. As theactivated amino groups of starting materials, for example, correspondingphosphoric amides can be used. As a method for removing (elimination) ofthe protected groups, for example, catalytic reduction in hydrogenstream in the presence of a catalyst of Pd-black or Pd-charcoal; acidtreatment with anhydrous hydrogen fluoride, methan sulfonic acid,trifluoromethan sulfonic acid, trfluoroacetic acid or their mixtures;base treatment with diisopropylethylamine, triethylamine, piperidine,piperadine or the like; or reduction with sodium in liquid ammonia canbe used. The elimination reaction by using the above acid treatment isconducted usually at a temperature of −20° C.˜40° C. In the acidtreatment, it is efficient to add a cation catching agent such asanisol, phenol, thioanisol, metacresol, paracresol, dimethylsulfide,1,4-butandithiol or 1,2-ethandithiol. The 2,4-dinitrophenyl group usedas an imidazole protection group of histidine is removed by thiophenoltreatment. The formyl group used as an indole protection group oftryptophan is removed by acid treatment in the presence of the above1,2-ethanedithiol, 1,4-butanedithiol or the like, and further it can beremoved by alkali treatment with a diluted sodium hydroxide solution,diluted ammmonia or the like.

The protection of functional groups that should not participate in thereaction of starting materials and the protected groups, elimination ofthe protected groups, activation of the functional group thatparticipate in the reaction or the like can be suitably selected fromwell-known groups and means. As the other methods for obtaining amidesof proteins, for example, an α-carboxyl group of carboxy end-group aminoacid is protected by amidation, a peptide chain (protein) is elongatedto the desired chain length at the side of the amino chain, a proteinthat the protected group of the α-amino group at the N-end of thepeptide chain is eliminated and a protein that the protected group ofthe carboxyl group at the C-end of the peptide are produced, and theboth peptide are condensed in a mixture solution as described above. Theparticular of the condensation reaction is as mentioned in the above.The protected protein obtained by the condensation is purified, allprotected groups are removed by the above method, and crude protein canbe obtained. The crude protein is purified by using a known purificationmeans, main fructions are lyophilized and an desired amide of theprotein can be obtained. For obtaining esters of the protein, forexample, α-carboxyl groups of carboxy-end amino acids are condensed withdesired alcohols to obtain amino acid esters, the esters are treated asshown in amides of the protein, and desired esters of the protein can beobtained.

The partial peptide or the salts of this invention can be produced bywell-known methods for synthesizing peptides or by cutting the proteinof this invention with a suitable peptidase. As the method forsynthesizing the peptide, for example, a solid-phase synthesis mathod orliquid-phase synthesis method can be used. Namely, a partial peptideable to constitute the partial peptide of this invention or an aminoacid is condensed with the remaining parts, when the product has aprotected group, the protected group is eliminated, and the desiredpeptide can be produced.

As well-known condensation methods and the elimination of the protectedgroups, for example, the following methods are exemplified. M. Bodanszkyand M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York(1966); Schroeder and Luebke, The Peptide, Academic Press, New York(1965); Nobuo Izumiya et al., Fundament and Experiments of PeptideSynthesis, Maruzen Co., (1975); Haruaki Yajima and Shunpei Sakakibara,Biochemical Experiment Lectures 1, Chemistry of Proteins IV, 205 (1977);Haruaki Yajima supervised, Continued Development of Medicines, Vol. 14,Peptide Synthesis, Hirokawa Shoten. After the reaction, further, thepartial peptide of this invention can be purified by usual purificationmethods, for example, solvent extraction, distillation, columnchromatography, liquid chromatography, recrystalization and theircombination. When the partial peptide is obtained by the above methods,it can be changed into a suitable salt by a known method or the similarmethod. When a salt of the partil peptide is obtained, it can be changedinto a released compound or the other salt by a known method or thesimilar method.

As the DNA coding the protein of this invention, any compounds that abase sequence coding the above-mentioned protein of this invention iscontained can be used. Further. a genome DNA, a genome DNA library, theabove-mentioned cDNA derived from cells and tissues, the above-mentionedcDNA library derived from cells and tissues, or synthetic DNA can beused. A vector used in the library can be selected from any one ofbacteriophage, plasmid, cosmid, phagemid and the like. Using a total RNAor mRNA fruction prepred from the above cells or tissues, it can bedirectly amplified by Reverse Transcriptase Polymerase Chain Reaction(called as a RT-PCR method hereinafter). As the DNA coding the proteinof this invention, any one of the following DNAs can be exemplified: forexample, a DNA containing a base sequence represented by SEQ ID No.2, ora DNA, which has a base sequence hybridizing with a base sequencerepresented by SEQ ID No.2 under highstringent conditions and codes theprotein having substantially the same activity as the protein of thisinvention (for example, citotoxin activity).

Further embodying, As the DNA coding a protein having the amino acidsequence represented by SEQ ID No. 1, the DNA having the base sequencerepresented by SEQ ID No. 2 can be used.

As the DNA coding the partial peptide of this invention, any one of DNAscontaining the above base sequence coding the partial peptide of thisinvention can be used. Further. a genome DNA, a genome DNA library, theabove-mentioned cDNA derived from cells and tissues, the above-mentionedcDNA library derived from cells and tissues, or synthetic DNA can beused. As the DNA coding the partial peptide of this invention, forexample, any one of the following DNAs can be exemplified: for example,a DNA having a partial sequence of DNA having a base sequencerepresented by SEQ ID No.2, or a DNA having a prtial sequence of DNA,which has a base sequence hybridizing with a base sequence representedby SEQ ID No.2 under highstringent conditions and codes the proteinhaving substantially the same activity as the protein of this invention(for example, citotoxin activity).

As cloning methods of the DNA perfectly coding the protein of thisinvention or the partial peptide (hereinafter, in the description of thecloning and expression of the DNA coding these protein and the like,case by case, these protein and the like is shorten as the protein ofthis invention), using a synthesized DNA primer having the partial basesequence of the protein of this invention, it is amplifyed by a knownPCR method, or the DNA combined in a suitable vector is selected byhybridization with a DNA fraction or a synthesized DNA that codes a partor all region of the protein of this invention. The hybridization methodcan be conducted by the description of, for example, Molecular Cloning,2^(nd), J. Sambrook et al., Cold Spring Harbor Lab. Press (1989). When acommertially available liblary is used, it can be conducted by themethod described in an attached explanation. In the change of DNAbasesequences, using a known kit such as MutanTM-G (produced by TakaraShuzou Co.), MutanTM-K (produced by Takara Shuzou Co.) or the like, aGapped duplex method, a Kunkel method, well-known methods or similarmethods can be conducted. The DNA coding a cloned protein can be used asit is, or by digesting with a restriction enzyme as desired, or byaddition of a linker. The DNA may have ATG, GTG or TTG as a translationinitiation codon at the 5′ end side and TAA, TGA or TAG as a translationtermination codon at the 3′ end side. The translation initiation codonand the translation termination codon can be added by using a suitablesynthetic DNA adapter. The expression vector of the protein of thisinvention can be produced, for example, by means (i) that an desired DNAis cutt off from the DNA coding the protein of this invention, and (ii)that the DNA fraction is bonded to the downstream of the promoter in asuitable expression vector.

As the vector, a plasmid derived from Echerichia coli (such as pBR322,pBR325, pUC12, pUC13 or pET30), a plasmid derived from Bacillus subtilis(such as pUB110, pTP5 or pC194), a plasmid (such as pSH19, pSH15),bacteriophage derived from yeast, a bacteriophage such as λ phage, ananimal virus such as a retro virus, a vaccinia virus, a Baculo virus orthe like, pA1-11, pXT1, pRc/CMV, pRc/RSV or pcDNAI/Neo can be used. Asthe promoter used in this invention, any promoters suitable for the hostused in the expression of genes can be used. For example, when animalcells are used as the host, a SR α promoter, SV40 early promoter,HIV•LTR promoter, CMV promoter or HSV-TK promoter can be exemplified. Inthese promoters, the CMV (cytomegalovirus) promoter, SR α promoter canbe preferably used. When the host is an Echerichia family fungus, a trppromoter, lac promoter, recA promoter, A PL promoter, lpp promoter or T7promoter is preferred. When the host is a Bacillus family fungus, a SPO1promoter, SPO2 promoter or penP promoter is preferred. When the host isyeast, a PHO5 promoter, PGK promoter, GAP promoter or ADH promoter ispreferred. When the host is an insect cell, a polyhedrin promoter, P10promoter or the like preferred.

As the expression vectors other than the above vectors, if desired,vectors containing an enhancer, a selection marker, SV40 replicationorigin (occationally abbreviated as SV40ori hereinafter) or the like canbe used. As the selection marker, for example, a dihydro folic acidreduction enzyme (occationally abbreviated as dhfr hereinafter) gene(methotrxate (MTX) resistance), an ampicillin resistance gene(occationally abbreviated as Ampr hereinafter), a neomycin resistancegene (occationally abbreviated as Neor hereinafter) G418 resistance andkanamycin resistance gene can be exemplified. Particularly, when thedhfr gene is used as a selection marker by using Chinese hamster cellsdefective a dhfr gene, combinant cells can be selected by a medium notcontaining thymidine. If necessary, further, a signal sequence fitted tothe host is added to N end sides of the protein of this invention. Whenthe host is an Echerichia family fungus, a PhoA signal sequence, OmpAsignal sequence or the like can be used. When the host is a Bacillusfamily fungus, an α-amylase signal sequence, a subtilicin signalsequence or the like can be used. When the host is yeast, an MF α signalsequence, a SUC2 signal sequence, a SUC2 signal sequence or the like canbe used. When the host is an animal cell, an insulin signal sequence, anα-interferon siganl sequence, an antibody molecular signal sequence orthe like can be used. Using a vector containing the DNA coding suchconstructed protein of this invention, a transformant can be produced.

As the host, for example, an Genus Echerichia, a Genus Bacillus, yeast,insect cells, insects, animal cells or the like can be used. As anembodiment of Genus Echerichia, for example, Escherichia coli K12, DH1,DH5α (Proc. Natl. Acad. Sci. USA, Vol. 60, 160(1968)), JM103 (NucleicAcids Research, Vol. 9, 309(1981)), JA221 (Journal of Molecular Biology,Vol. 120, 517(1978)), HB101 (Journal of Molecular Biology, Vol. 41,459(1969)), C600 (Genetics, Vol. 39, 440(1954)) or the like can be used.As Genus Bacillus, for example, Bacillus subtilis MI114 (Gene, Vol. 24,255(1983), 207-21 (Journal of Biochemistry, Vol. 95, 87(1984)) or thelike can be used. As the yeast, for example, Saccharomyces cerevisiaeAH22, AH22R, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombeNCYC1913, NCYC2036, Pichia pastoris KM71 or the like can be used.

As the insect cells, for example, when the virus is AcNPV, Spodopterafrugiperda cell; Sf cells, MG1 cells derived from midintestine ofTrichoplusia ni, High Five TM cells derived from the egg of Trichoplusiani, cells derived from Mamestra brassicae, cells derived from Estigmenaacrea or the like can be used. When the virus is BmNPV, Bombyx mori Ncells; BmN cells or the like can be used. As the Sf cells, for example,Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J. L. et al., In Vivo, 13,213-217(1977)) or the like can be used. As the insects, for example,larvae of silkworms can be used (Maeda et al, Nature, Vol. 315,592(1985)). As the animal cells, for example, monkey cell COS-7, Vero,chinese hamster cell CHO (abbreviated as a CHO cell hereinafter), dhfrgene defective chainese hamstercell CHO (abbreviated as a CHO(dhfr-)cell hereinafter), mouse L cells, mouse AtT-20, mouse myeloma cells, ratGH3, human FL cells or the like can be used. Moreover, several kinds ofnormal human cells, for example, liver cells, splenocytes, nerve cells,neuroglia, spleen β cells, bone marrow cells, mesangium cells,Langerhans cells, skin cells, epithelium cells, endothelium, fibroblast,fibrocells, muscle cells, fat cells, immunological cells (for example,macrophages, T cells, B cells, natural killer cells, mast cells,neutrophils, basophils, eosinophils, monocytes), megakaryocytes,synovial cells, cartilages, bone cells, osteoblasts, osteclasts, mammarygrand cells, hepatic cells, interstitial cells, or precursor cells, stemcells or cancer cells of these cells can be used. For the transformationof Genus Echerichia, for example, it can be conducted by the methoddescribed in Proc. Natl. Acad. Sci. USA, Vol. 69, 2110(1972), Gene, Vol.17, 107(1982) or the like.

For the transformation of Genus Bacillus, for example, it can beconducted by the method described in Molecular & General Genetics, Vol.168, 111(1979) or the like. For the transformation of yeast, forexample, it can be conducted by the method described in Methods inEnzymology, Vol. 194, 182-187(1991), Proc. Natl. Acad. Sci. USA, Vol.75, 1929(1978) or the like. For the transformation of insect cells orinsects, for example, it can be conducted by the method described inBio/Technology, 6, 47-55(1988) or the like.

For the transformation of animal cells, for example, it can be conductedby the method described in Cell Engineering, separate volume 8, New Cellengineering Experiment protocol, 263-267(1995), published by Shujunsha,and Virology, Vol. 52, 456 (1973). Using the above methods,transformants, which are transformed with expression vectors containingDNA coding the protein, can be obtained. When the transformants that thehosts are Genus Echerichia or Genus Bacillus are cultured, a liquidmedium is suitable as the medium used in the culture. In the medium,carbon sources, nitrogen sources, inorganic and the like, which arenecessary for the growing of the transformants, are contained. As thecarbon sources, for example, glucose, dexitrin, soluble starch, succroseor the like can be exemplified. As the nitrogen sources, for example,inorganic or organic materials such as ammonium salts, nitrates, cornsteep liquor, peptone, casein, meat extract, bean cakes, potato extractand the like can be used. As the inorganic materials, for example,calcium chloride, monosodium phosphate and magnesium chloride can beexemplified. Yeast extract, vitamines, growth-stimulating materials orthe like can be added. About pH 5-8 of the medium is preferrably used.

As the medium for culturing Genus Escherichia, for example, a M9 mediumcontaining glucose and casamino acid (Miller, Journal of Experiments inMolecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York1972) is preferred. If necessary, for efficient action of the promoter,for example, an agent such as 3β-indolyl acrylic acid can be added. Whenthe host is Genus Escherichia, the culture is usually concducted at atemperature of about 15˜43° C. for about 31˜24 hours, if necessary,aeration or stirring can be added. When the host is Bacillus familyfungi, the culture is usually conducted at a temperature of about 30˜40°C. for about 6˜24 hous, if necessary, aeration or stirring can be added.

When the transformant that the host is yeast is cultured, as the medium,for example, a Burkholder minimal medium (Bostian, K. L. et al., Proc.Natl. Acad. Sci. USA, Vol. 77, 4505(1980)) or a SD medium containing0.5% casamino acid (Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA,Vol. 81, 5330(1984)) can be exemplified. About pH 5-8 of the medium ispreferrably used. The culture is usually conducted at a temperature ofabout 20˜35° C. for about 24˜72 hours, if necessary, aeration orstirring can be added. When the transformant that the host is an insectcell or an insect is cultured, as the medium, Grace's Insect Medium(Grace, T. C. C., Nature, 195,788(1962)) can be used by suitably addingan addition of immobilized 10% bovine serum or the like. About pH6.2-5.4 of the medium is preferrably used. The culture is usuallyconducted at a temperature of about 27° C. for about 3˜5 days, ifnecessary, aeration or stirring can be added. When the transformant thatthe host is an animal cell, as the medium, for example, a MEM mediumcontaining about 5˜20% fetus bovine serum (Science, Vol. 122,501(1952)), a D M E M medium (Virology, Vol. 8, 396(1959)), an RPMI 1640medium (The Jounal of the American Medical Association, Vol. 199,519(1967)), a 199 medium (Proceeding of the Society for the BiologicalMedicine, Vol. 73,1(1950)) or the like can be used. About pH 6-8 of themedium is preferrably used. The culture is usually conducted at atemperature of about 30˜40° C. for about 15˜60 hours, if necessary,aeration or stirring can be added. As descrived in the above, theprotein of this invention can be produced out of the cells of thetransformants.

For separation and purification of the protein of theis invention, forexample, the following methods can be suitably used. When the protein ofthis invention is extracted from the cultured fungi or cells, after theculture, the fungi or cells are collected by a well-known method,suspended in a suitable buffer, and destroyed by ultrasound, lysozymeand/or freze-thawing or the like. Then, the crude extracted liquid ofthe protein is obtained by centrifugation or filtration. A proteindenaturant such as urea or guanidine hydrochloride, or a surface-activeagent such as tritonX-100TM can be contained in the buffer. When theprotein is secreted in the medium liquid, after the culture is finished,the fungi or cells and supernatant are separated by a well-known method,and the supernatant is collected. Such obtained culture supernatant orthe protein contained in the extracted liquid is purified by acombination of well-known separation and purification methods. Thesewell-known separation and purification methods are methods usingsalting-out techniques or solubilities by a solvent precipitationmethod, a dialysis method, a method using a difference in the molecuarweight such as ultrafiltration and SDS-polyacrylamide gelelectrophoresis or the like, a method using a difference of the chargesuch as ion exchange chromatography, a method using a difference ofhydrophobic such as hydrophobic chromatography, a method using specificaffinity such as afnity chromatography, a method using a difference ofhydrophobic such as reverse phase high-speed liquid chromatography, amethod using a difference of isoelectric points such asisoelectrcophoresis can be used.

When such a protein is obtained in the form of free, it can be convertedinto a salt by a well-known method or a similar method. On the otherhand, when the protein is obtained in the form of a salt, it can beconverted into a free form or the other salt by a well-known method or asimilar method. The protein produced by a recombinant may be furtherreacted with a suitable protein modification enzyme before or afterpurifying the protein to optionally modify or partially remove thepolypeptide. As the protein modification enzyme, for example, trypsin,chymotrypsin, arginylendopeptidase, protein kinase, glycosidase or thelike may be used. The activitis of such obtained protein of thisinvention or salts thereof may be determined by a bond experiment with alabeled ligand and enzyme immunoassay using a specific antibody or thelike.

The antibodies for the protein, the partial peptide or the salts of thisinvention, may be a polyclonal antibody or monoclonal antibody oncondition that the antibodies can recognize the protein, the partialpeptide and the salts. The antibodies for the protein, the partialpeptide or the salts of this invention (in the following description ofantibodies, these protein and the like may be abbreviated as the proteinof this invention) may be produced by using the protein as the antigenand by a well-known production method of antibodies or anti-serum.

Production of a Monoclonal Antibody

-   (a) Production of cells of monoclonal antibody production: The    protein of this invention is administered to a warm-blodded animal    at a position, which can produce an antibody by the administration,    with itself or a carrier or a diluent. In the administration, to    enhance the effect of antibody production, a complete Freund's    adjuvant or an incomplete Freund's adjuvant may be administered. The    administration is usually conducted once for each 2˜6 weeks, and    total is 2˜10 times. The warm-blooded animals are, for example,    monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats,    chickens and the like, preferably mice and rats. At the production    of cells for producing a monoclonal antibody, the warm-blooded    animal immunized with an antigen, for example, mice are used. A    mouse recognizable an antibody value is selected from the mice, the    spleen or the lymph is collected after 2˜5 days of the final    immunization, and the cells of monoclonal antibody production    contained in the spleen or the lymph are fused with myeloma cells of    the same kind or different kind of animals to prepare a hybridoma    for producing a monoclonal antibody. The antibody value in the    antiserum may be determined, for example, by a method that the    antiserum is reacted with an after-mentioned labeled protein, and    the activity of the labeled agent bound to the antibody is measured.    The fusion operation may be conducted by a known method such as a    method of Kohler and Milstein (Nature, 256, 495 (1975)). As the    fusion promotion agent, for example, polyethylene glycol (PEG), a    Sendai virus or the like, preferably PEG may be used.

As the myeloma, for example, myloma celles of warm-blooded animals suchas NS-1, P3U1, SP2/0, AP-1 and AP-1, preferably P3U1 can be exemplified.The preferabl ratio of the number of the antibody production cells(spleen cells) to the number of myeloma cells is around 1:1˜20:1. PEG(preferably, PEG1000-PEG6000) is added in the concentration of around10˜80%, and incubation is conducted at a temperature of 20˜40° C.,preferably 30˜37° C. for 1˜10 minutes, and cell fusion is efficientlyperformed. In screening of a hybridoma of monoclonal antibodyproduction, several kinds of methods can be used, for example, there area method that a supernatant of hyblidoma culture is added to a solidphase (such as a micro plate) adsorbing a protein antigen directly orwith a carrier, and an anti-immunoglobulin antibody, which is labeledwith a radioactive material or an enzyme yeast, (when the cells forusing in cell fusion is a mouse, an anti-mouse immunoglobulin is used)or a protein A is added so as to detect the monoclonal antibody bound tothe solid phase; and a method that a supernatant of hyblidoma culture isadded to a solid phase adsorbing an anti-immunoglobulin antibody or aprotein A, a protein labeling a radioactive material or an enzyme yeastis added, and the monoclonal antibody bound to the solid phase isdetected. The selection of the monoclonal antibody may be conducted by aknown method or a similar method thereof. Usually, it may be conductedby a medium for animal cells that HAT (hypoxanthine, amino pterin,thymidine) is added. As the medium for selection and breeding, anymedium that the hyblidoma can breed may be used. As an example, a RPMI1640 medium containing 1˜20%, preferably 10˜20% bovine fetus serum, aGIT medium containing 1˜10% bovine fetus serum (produced by Wako JunyakuKougyou Co.) or a medium not containing serum for hybridoma culture(SFM-101, Nissui Seiyaku Co.) may be used. The culture temperature isusually 20˜40° C., preferably about 37° C. The culture time is usually 5days˜3 weeks, preferably 1 week˜2 weeks. The culture may be usuallyconducted in a 5% carbon dioxide. The antibody value of the supernatantof hyblidoma culture can be determined as shown in the above-mentionedmethod for measuring the antibody value in the antiserum.

-   (b) Purification of the monoclonal antibody: The separation and    purification of the monoclonal antibody may be conducted by a known    method, for example, a separation and purification method for    immunogloblins (such as a salting-out method, an alcohol    precipitation method, a isoelectric precipitation method, an    electrophoresis method, a adsorption and desorption method with an    ion-exchanger (for example, DEAE), a ultracentrifuge method, a gel    filtration method, or a specific purification method that only an    antibody is cllected with an active absorbant such as an    antigen-binding solid phase or protein A or protein G, and the bond    is released to obtain the antibody).

Production of the Polyclonal Antibody

The polyclonal antibody of this invention can be produced by a knownmethod or a similar method. As an example, using an immunoanigen(protein antigen) itself or a complex of the immunoantigen and a carrierprotein, it immunizes to a warm-blooded animal by using the same methodas in the above-mentioed method for producing a monoclonal antibody,materials containing the antibody for the protein of this invention arecollected, and the antibody is separated and purified. As to the complexof an immunoantigen for immunizing a warm-blooded animal and a carrierprotein, the kind of the carrier protein and a mixture ratio of thecarrier to a hapten are not important, on condition that the antibodycan be efficiently produced for the hapten immunized by closslinkingwith the carrier. For example, the bovine serum albumin, bovinecyclogloblin, hemocyanin or the like can be used for coupling with thehapten at a weight ratio of about 0.1˜20, preferably about 1˜5 to 1 ofhapten. In the coupling of the hapten and the carrier, several kinds ofcondensation agents such as active ester agents containingglutaraldehyde, carbodiimide, maleimid active ester, a thiol group and adithiopyridyl group can be used. The products from the condensation areadministered to the parts of warm-blooded animals able to produce theantibody, along with itself or a carrier or a diluent. To enhance theability of antibody production at the administration, a completeFreund's adjuvant or incomplete Freund's adjuvant may be administered.The administration is usually conducted once for every 2˜6 weeks, andtotal is about 3˜10 times. The polyclonal antibody can be collected fromblood, ascites, preferably blood of the warm-blooded animal immunized bythe above method. The polyclonal antibody value in the antiserum isdetermined by the same method described in the above determination ofthe antibody value in the antiserum. The separation and purification ofthe polyclonal antibody can be conducted by the same method ofseparation and purification of immunogloblin as in that of the aboveseparation and purification of the monoclonal antibody.

The treating agents containing the protein or the partial peptide ofthis invention and the protein of this invention and the like havecancer cytotoxic activity, so that the agents may be used as agents forextracting disease tissues (the extract contains all and partial,preferably partial extract), and particularly, for treating fixedcancer. When the protein of this invention and the like is used as theabove treating and preventing agents, it is used after it is purified toat least 90%, preferably 95% or more, more preferably 98% or more, andfurther preferably 99% or more.

The inhibition activity of cancer cell proliferation of the protein ofthis invention or the like can be determined by a known method, or acytotoxin activity or an activity causing cell death is determined byknown method or a similar method, preferably by the method described inthe after-mentioed experiments. As the test compounds, for example,peptides, proteins, non-peptide compounds, synthetic compounds,fermentation products, cell extract, plant extract, animal tissueextract and blood plasma can be exemplified. These compounds may be newcompounds or known compounds.

The compounds or the salts obtained by the screening method or the kitfor screening of this invention are selected from the above testcompounds, for example, peptides, proteins, non-peptide compounds,synthetic compounds, fermentation products, cell extract, plant extract,animal tissue extract and blood plasma. These compounds have an activityinhibiting the cytotoxic activity of the protein of this invention orthe like or the inhibition activity of cancer cell proliferation of theprotein of this invention or the like. As the salts of the compounds,the salts similar to the salts of the protein of this invention can beused.

When the compounds obtained by using the screening method or the kit forscreening are used as the above treating agents, they can be used byusual means. For example, they are used as tablets, capsules, elixirs,microcapsules, sterile solutions, suspension or the like. As suchobtained pharmaceutical preparations are safety and have low toxicity,for example, they may be administered to a human or a warm-bloodedanimal (such as mice, rats, rabbits, goats, pigs, cows, horses, birds,cats, dogs, monkeys and the like). The dosage weights of the compoundsor salts thereof are changed by the action, the disease, the dosageroute or the like. Commonly, in adults (estimated at 60 kg weight),about 0.1˜100 mg of the compound per day, preferably about 1.0˜50 mg,more preferbly about 1.0˜20 mg can be administered. In parenteraladministration, the dosage of the compounds that is variable by theobject, the disease and the like, usually in adults (estimated at 60 kgweight), is suitably about 0.01˜30 mg of the compound per day, morepreferably about 0.1˜20 mg, further preferably about 0.1˜10 mg byintravenous injection. In the other animals, the dosage of the weightestimated at 60 kg can be used.

Screening of the candidate compounds of medicine for diseases: Since theprotein or the like of this invention have a cytotoxic activity, thecompounds or the salts promoting the function (such as a cytotoxicactivity) of the protein or the like of this invention, for example, maybe used as agents for treating cancers. On the other hand, the saltsinhibiting the function of the protein or the like of this invention,for example, may be used as agents for treating and preventing gastritisand gastric ulcer. Accordingly, the protein or the like of thisinvention are effectively used as reagents for screening the compoundsor the salts that promote or inhibit the function of the protein or thelike of this invention.

Namely, this invention provides (1) a screening method characterized inthat the protein or the partial peptide or the salts of this inventionare used, and the method screens the compounds promoting the function(such as a cytotoxic activity) of the protein or the partial peptide orthe salts of this invention, or the method screens the compoundsinhibiting the function (such as a cytotoxic activity) of the protein orthe partial peptide or the salts of this invention. The compoundspromoting the function may be abbriviated as ‘promoters’ and thecompounds inhibiting the function is abbreviated as ‘inhibitors’hereinafter.

Moreover, this invention provides (2) a kit for screening promoters orinhibitors, characterized in that the protein or the prtial peptide orthe salts of this invention are contained. The above kit may beabbreviated as ‘a kit for screening of this invention’ hereinafter.

In embodiment, for example, in the above (1), it provides a screeningmethod of promoters or inhibitors, characterixed in that comparison isconducted between case (i) and case (ii). The case (i) is that theprotein or the partial peptide or the salts of this invention arecontacted with a cell, which is a normal cell containing a blood cellderived from a tissue of the above warm-blooded animals (preferablyhuman) or the above-mentioned cancer cell. The case (ii) is that theprotein or the partial peptide or the salts of this invention arecontacted with a cell, which is a normal cell containing a blood cellderived from a tissue of the above warm-blooded animals (preferablyhuman) or the above-mentioned cancer cell, and a test compound.

In embodiment, further, in the above (2), it provides a kit forscreening promoters or inhibitors, characterixed in that it contains theprotein or the prtial peptide or the salts of this invention, and a cellthat is a normal cell containing a blood cell derived from a tissue ofthe above warm-blooded animals (preferably human) or the above-mentionedcancer cell or the like.

Further, concretely, in the screening method, case (i) and case (ii) arecharacterized in that the cytotoxic activities of the protein and thelike of this invention are determined and compared.

The cytotoxic activity, the cell multiplication inhibition activity, andthe activity causing cell death of the protein or the like of thisinvention can be determined by a known method or a similar method.However, more concretely, using established cell lines and the like,further, a substrate containing the test compound, a negative controlwhich is a substrate not containing the test compound, and a positivecontrol which is a substrate containing M toxin, these three or two areused. Comparing the cell numbers under the conditions satisfyable instatistical significance, inhibition activity of the cytotoxic activityor the cell multiplication activity, or the specified sample having theinhibition activity of the cytotoxic activity or the cell multiplicationactivity can be detected by existence or absence of the activities orincrease and decrease. The cells used in the detection method are, forexample, normal cells containing a blood cell derived from a tissue ofthe above warm-blooded animals (preferably human) or the above-mentionedcancer cells of several kinds of warm-blooded animals (for example,cancer of the endometrium, endometrioma, breast cancer, cancer of thestomach, hepatic carcinoma, spleen cancer, carcinoma of the galbladder,colon cancer, prostatic cancer, lung cancer, renal cancer,neuroblastoma, cancer of the urinary bladdar, malignant melanoma, cancerof the tongue, carcinoma of the gingiva, mouse fibroblast, African greenmonkey kidneys rat liver cancer, and the like).

As the test compunds, for example, peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cell extracts,plant extracts, animal tissue extracts and the like can be exemplified.These compounds may be new compounds or known compounds. For conductingthe above screening method, the protein or the like of this invention issuspended in buffer suitable to the screening, and the samples of theprotein or the like of this invention are prepared. As the buffer,phosphate buffer of pH about 4˜10 (preferably, pH about 6˜8),Tris-hydrochloride buffer or the like, which does not inhibit thereaction of the protein or the like of this invention and the testcompounds, may be used.

As concrete screening method, after the screening examination, {circlearound (1)} a method for directly observing the cell changes under amicroscope and counting the cells with a hemocytometer or the like,{circle around (2)} a method that the change of potassium, hemoglobinsand the like, which are eluted from cells by the cell dath, is catched,{circle around (3)} a method for determining the remaining cells afterthe reaction with tetrazolium salts or the like, {circle around (4)} amethod for determining the remining living cells with a radioactivelabelled subsatance, {circle around (5)} a method for confirming thecell death by induction of cell apoptosis and the like can beexemplified. For example, as a compound increasing the cytotoxicactivity of the protein or the like of this invention, a test compoundin which the cytotoxic activity in the above case (ii) compared with theabove case (i) is increased by about 20% or more, more preferably 30% ormore, further preferably 50% or more, can be selected. On the otherhand, as a compound inhibiting the cytotoxic activity of the protein orthe like of this invention, a test compound in which the cytotoxicactivity in the above case (ii) compared with the above case (i) isinhibited by about 20% or more, more preferably 30% or more, furtherpreferably 50% or more, can be selected. These may be conducted as amethod for high throughput screening. In the following, as method{circle around (2)}, a method for determining hemoglobins by hemolyticreaction and as method {circle around (3)}, a WST method arerespectively used. In the methods, active carbon, CM cellulose andcalcium alginate are selected as adsorbents which show the anti-M toxinactivity.

It is further possible to examine and compare the solutions containingthese anion control, positive control and test compounds with animalmodels to confirm the effects of the animal levels of the anti-M toxicmaterials. In these cases, many kinds of warm-blooded animals may beused. Particularly, a mouse, a rat, a dog, and a monkey may be used. Asinfected models, a Mongolian gerbil, a mouse and a monkey areeffectively used.

When the compounds obtained by the screening method or the kit forscreening of this invention are used as the above treating andpreventing agents, these compounds can be used by usual means. Forexample, using the same methods of the pharmaceutical preparations ofthe protein of the invention, they are used as tablets, capsules,elixirs, microcapsules, sterile solutions, suspension or the like. Assuch obtained preparations are safety and have low toxicity, forexample, they may be administered to a human or a warm-blooded animal(such as a mouse, a rat, a rabbit, a goat, a pig, a cow, a horse, abird, a cat, a dog, a monkey and the like). The dosage weights of thecompounds or salts thereof are changed by the action, the disease, thedosage route or the like. When the compounds are used for increasing thefunction of the protein or the like of this invention as a tissueregeneration agent after removing the disease tissues, commonly, inadults (estimated at 60 kg weight), about 0.1˜100 mg of the compound perday, preferably about 1.0˜50 mg, more preferbly about 1.0˜20 mg can beorally administered. For the other animals, the dosage of the weightestimated at 60 kg can be used.

Quantification of the protein or the partial peptide or the salts ofthis invention:

The antibodies for the protein and the like of this invention(occasionally, abbreviated as the antibodies of this invention,hereinafter) can specifically recognize the protein and the like of thisinvention, so that they can be used for quantification of the proteinand the like of this invention in test liquid, particularly,quantification by a sandwich immunization technique. Namely, thisinvention provides (i) a method for quantifying the protein and the likeof this invention in test liquid, characterized in that the antibody ofthis invention and test liquid and the protein and the like of thisinvention are conpetitively reacted, the ratios of the labelled proteinand the like of this invention that bind to the antibody are determined,and (ii) a method for quantifying the protein and the like of thisinvention in test liquid, characterized in that the test liquid and theantibody insolubilized on a carrier and the other labelled antibody ofthis invention are reacted at the same time or continuously, and thenthe activity of the labeling agent on the insolubilized carrier isdetermined. In the above quantitative method (ii), preferably, oneantibody is an antibody recognizing the N ends of the protein and thelike of this invention, the other antibody is an antibody reacting withthe C ends of the protein and the like of this invention.

Moreover, using a monoclonal antibody for the protein and the like ofthis invention (ocasionally abbreviated as a monoclonal antibody of thisinvention, hereinafter), the quantification of the protein and the likeof this invention can be conducted, and further, the detection can beconducted by using tissue dyeing. As these objects, antibody molecularsthemselves may be used, or F(ab′)2, Fab′, or Fab fraction of antibodymoleculars may be used. The quantification of the protein and the likeof this invention using of this invention should not be limited. Forexample, the quantity of the antibody, antigen or antibody-antigencomplex corresponding to the quantity of the antigen (for example, thequantity of the protein) in the test liquid is chemically or physicallydetected, the obtained quantity is determined by a standard curve thatis formed by standard liquid containing the antigen of a known quantity.As examples, nephelometry, a competition method, an immunometric method,and a sandwich method are preferably usable. Considering sensitivity andspecificity, the after-mentioned sandwich method is preferred. As thelabelling agents used in a determination method with a labelledsubstance, for example, a radioactive isotope, enzyme, fluorescencesubstance, luminescent substance and the like are exemplified. As theadioactive isotope, for example,

¹²⁵ I

,

¹³¹I

,

³H

,

¹⁴C

may be used. As the enzyme, stable and high active isotopes, forexample, β-galactosidase, β-glucosidase, alkaliphosphatase, peroxidase,a dehydrogenase of malic acid and the like may be used. As thefluorescence substance, for example, fluorescamine, fluoresceinisothiocyanate and the like may be used. As the luminescent substance,for example, luminol, luminol derivatives, luciferin, lucigenin and thelike may be used. Moreover, for the bind of an antibody or an antigenand a labelling agent, a biotin-avidin type may be used.

The insolubilization of an antigen or an antibody may be conducted byphysical adsorption, or the chemical bonding usually used for theinsolubilization of proteins or enzymes. As the carrier, insolublepolysaccharides such as agarose, dextran and cellulose, synthetic resinssuch as polystyrene, polyacrylamide and silicone or glass can beexemplified. In the sandwich method, the test liquid is reacted with theinsolubilized monoclonal antibody of this invention (primary reaction),then it is reacted with the other labelled monoclonal antibody of thisinevention (secondary reaction), and the activity of the labelling agenton the insolubilized carrier is determined to determine the quantity ofthe protein of this invention in the test liquid. The primary reactionand the secondary reaction can be changed. Otherwise these reactions maybe conducted at the same time or by sliding the starting times. Thelabelling agent and the isolubilizing method may be treated the same asthese reactions. In the immuno assay by using the sandwich method, it isunnecessary that the antibody used as an antibody for a solid phase oran antibody for labelling is one kind. A mixture of two or more kinds ofantibodies may be used for improving the determination sensitivity. Inthe determination method of the protein and the like of this inventionby using the sandwich method of this invention, the monoclonalantibodies used in the primary reaction and the secondary reactionpreferably have different parts that the protein and the like of thisinvention is bound. Namely, as to the antibodies used in the primaryreaction and the secondary reaction, for example, when the antibody usedin the secondary reaction recognaize the C end of the protein and thelike of this invention, the antibody used in the primary reactionpreferably recognize a part except the C end, for example, the N end.

The monoclonal antibody of this invention may be used in a determinationsystem except the sandwich method such as a competition method, or animmunometric method or nephelometry. In the competition method, antigensand labelled antigens in test liquid are competitively reacted withantibodies, and then unreacted labelled antibodies (F) and labelledantigens (B) bound to the antibodies are separated (B/F separation), thelabelled quantity of B or F is determined, and the antigen quantity isquantified. In the reaction method, soluble antibodies are used asantibodies. In the B/F separation, a liquid phase method thatpolyethyleneglycol or the second antibody for the above antibody isused, and a solid phase method that a solid phase antibody is used asthe first antibody, or a soluble antibody is used as the first antibodyand a solid phase antibody is used as the second antibody areexemplified. In the immunometric method, the antigens and the solidphase antigens in the test liquid are competitively reacted withlabelled antibodies of a definite amount and then the solid phase andthe liquid pahse are separated. Otherwise, the antigens in the testliquid and the labelled antibodies of an excess amount are reacted, thesolid phase antibodies are added to bind the unreacted labelledantibodies to the solid phase, and the solid phase and the liquid phaseare separated. Continuously, the labelled quantity of any one of theboth phases is determined, and the antigen quantity in the test liquidis determined. In the nephelometry, the quantity of unsolubleprecipitates occured as the result of the antigen-antibody reaction inthe gel or in the solution is determained. When the quantity of antigensin the test liquid is little and a small amount of precipitates areobtained, lasernephelometry using laser scattering and the like may bepreferably used.

When these immunological determination methods are applied in thedetermination method of this invention, special conditions andestablishment of operations are unnecessary Adding common technicalideas in the fields to usual conditions and operation methods in eachmethod, the determination systems of the protein and the like of thisinvention may be constructed. As to the details of these commontechnical means, general books, specialized books may be referenced. Forexample, Kan Irie edited, Radioimmuno Assay, Kodan-sha (1974), Kan Irieedited, Radioimmuno Assay, continued, Kodan-sha (1979), Eiji Ishikawa etal. edited, Immunoenzymometric Assay, Igaku Shoin (1978), Eiji Ishikawaet al. edited, Immunoenzymometric Assay, 2^(nd) edition, Igaku Shoin(1982), Eiji Ishikawa et al. edited, Immunoenzymometric Assay, 3rdedition, Igaku Shoin (1987), Methods in ENZYMOLOGY, Vol. 70,Immunochemical Techniques (Part A): ibid., Vol. 73, ImmunochemicalTechniques (Part B), ibid., Vol. 74, Immunochemical Techniques (Part C),ibid., Vol. 84 (Immunochemical Techniques (Part D: SelectedImmunoassays), ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods), ibid., Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies), all are published by Academic Press Co. can be referenced.As described above, by using the antibody of this invention, the proteinand the like of this invention can be sensitively quantified. Moreover,by quantifying the concentrations of the protein and the like of thisinvention by using the antibody of this invention, when the incresedconcentrations of the protein and the like of this invention aredetected in patients of Helicobacter pylori infection, patients havediseases, for example, gastritis, gastric ulcer, gastric cancer,valvular disease, diabetes mellitus, various cancers (such as cancer ofthe endometrium, endometrioma, breast cancer, colon cancer, prostaticcancer, lung cancer, hepatic carcinoma, spleen cancer, carcinoma of thegalbladder, renal cancer, neuroblastoma, cancer of the bladdar,malignant melanoma and the like). Otherwise, it is possible to diagnosethat the possibility of future morbidity is high. The antibody of thisinvention can be used for detection of the protein and the like of thisinvention in test liquid such as body fluid or tissues. It is used forformation of the antibody column for using purifying the protein and thelike of this invention, detection of the protein and the like of thisinvention in each fraction at the purification, and analysis of thebehavior of the protein and the like of this invention in test cells.

The medicine containing the antibody of this invention, the antibody ofthis invention (neutralizing antibody) having an action for neutralizingthe activities of the protein and the like of this invention can be usedas medicines for treating and preventing diseases such as gastritis,gastric ulcer, gastric cancer, valvular disease, diabetes mellitus,various cancers (such as cancer of the endometrium, endometrioma, breastcancer, colon cancer, prostatic cancer, lung cancer, renal carcinoma,neuroblastoma, cancer of the bladdar, malignant melanoma and the like).The humanized antibody of this invention for the protein and the like ofthis invention can be used as medicines for treating and preventingdiseases such as gastritis, gastric ulcer, gastric cancer, valvulardisease, diabetes mellitus, various cancers (such as cancer of theendometrium, endometrioma, breast cancer, colon cancer, prostaticcancer, lung cancer, renal carcinoma, neuroblastoma, cancer of thebladdar, malignant melanoma and the like). The humanized antibody can beformed with reference to the methods described in Nat Biotechnol, 14,845-851 (1996), Nat Genet. 15, 146-156 (1997) and PNAS, 97(2), 722-727(2000). In the following, these neutralizing antibody and humanizedantibody of this invention are abbreviated as the antibody of thisinvention.

The above treating and preventing agents containing the antibody of thisinvention can can be orally or parenterally administered as liquid as itis or a medicine composition of a suitable dosage form to human ormammalia (such as a mouse, a rabbit, a goat, a pig, a cow, a cat, a dog,a monkey and the like). The dosage of the agents is changed by theobject, the disease, the condition of illness, the dosage route or thelike. When the agents are used for treating or preventing a tumor of theendometrium, a dose of the antibody of this invention is usually 0.01˜20mg/kg weight, preferably 0.1˜10 mg/kg weight, more preferably 0.1˜5mg/kg weight about 1-5 times per day, preferably about 1-3 times perday. It is conveniant to administer the agent by an intravenousinjection. A dosage in the other parenteral or oral administration alsomay be according to the above dosage. When the condition of illness isvery severe, the dosage may be increased in proportion to the condition.The antibody of this invention can be administered as it is or as asuitable medical composition. The medical composition used in theadministration contains a pharmacologically allowable carrier for theabove antibody or the salt, a diluent or an excipient. Such acomposition may be provided as a dosage form suitable for oral orparenteral administration. Namely, for example, as the composition fororal administration, the dosage form is solid or liquid, concretely atablet (containing a sugar-coated tablet and a film coated tablet), apill, a granule, powder, a capsule (containing a soft capsule), syrup,emulsion, suspension or the like. Such a composition is prepared by aknown method, and it may contain the carrier usually used, a diluent oran excipient. For example, as the carrier for the tablet and theexcipient, lactose, starch, sucrose, magnesium stearate and the like canbe used.

After-mentioned sequence numbers of the sequence listing show thefollowing sequence.

SEQ ID No.1: it shows an amino acid sequence deribed from Helicobacterpylori 60190 (M toxin).

SEQ ID No.2: it shows a base sequence of DNA that codes derived fromHelicobacter pylori 60190 protein (M toxin) of this invention having theamino acid sequence represented by SEQ ID No.1.

SEQ ID No.3: it shows a base sequence of the primer (synthetic) DNA usedin Example 3.

SEQ ID No.4: it shows a base sequence of the primer (synthetic) DNA usedin Example 3.

The transformant, Escherichia coli M toxin/pET30EK/LIC/DH5α obtained inafter-mentioned Example 3 has been deposited by deposit No. FERM BP-8218on 17 Oct. 2002 at the International Patent Organism Depository,National Institute of Advanced Industrial Science and Technology (IPOD),AAIST, Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba City, IbarakiPrefecture, 305-8516, Japan. Moreover, the hybridoma clone No. 4obtained in after-mentioned Example 4 has been deposited by deposit No.FERM BP-8222 as BALB-c/P3U1/004-1G9 on 23 Oct. 2002 at the InternationalPatent Organism Depository, National Institute of Advanced Industrialand Technology (IPOD). Further, the hybridoma clone No. 101 has beendeposited by deposit No. FERM BP-8223 as BALB-c/P3U1/101-1C10 on 23 Oct.2002 at the International Patent Organism Depository, National Instituteof Advanced Industrial Science and Technology (IPOD) . The hybridomaclone No. 116 has been deposited by deposit No. FERM BP-8224 asBALB-c/P3U1/116-5D7 on 23 Oct. 2002 at the International Patent OrganismDepository, National Institute of Advanced Industrial Science andTechnology (IPOD).

EXAMPLES

This invention will be understood more readily in reference to thefollowing examples. However, these examples are intended to illustratethis invention and are not to be construed to limit the scope of theinvention. The gene manipulation using E. coli was according to themethod described in Molecular Cloning.

Example 1 A Method for Purifying and Extracting the Toxin of thisInvention from Helicobacter pylori

Helicobacter pylori can be obtained by a separated strain which hasalready established (for example, from American Type Culture Collection)or by culturing a strain separated from clinical test samples. In thisexample, a separated strain of Helicobacter pylori 60190 was used. Usingan agar medium that 5% bovine serum (produced by Sigma Co.) was added toa Brain Heart Infusion agar medium (poduced by Difco Co.), thisseparated strain was subcultured in 2-5 passages for about 1-2 weeks ata temperature of 37° C. and a humidity of 90% or more under microaerobicconditions (CO₂ 5-10%). It was confirmed under a microscope that thecells were not died or not in a colloi form but grown satisfactorily.The cells were transferred to Brain Heart Infusion agar culture producedby Difco Co.) plate containing not serum but 5%2,6-di-O-methyl-β-cyclodextrin. After the culture and grown conditionswere confirmed under the same conditions as described in the above, thecells were transferred to cultures containing2,6-di-O-methyl-β-cyclodextrin with gradual stepwise decreases, i.e.,2%, 1% and 0.5% of the concentration.

In a liquid culture of Brain Heart Infusion containing 0.5%2,6-di-O-methyl-β-cyclodextrin, the cells were cultured at a temperatureof 37° C. under microaerobic conditions for about 16 hours, whileagitated with a rotary shaker at 100-120 rpm. Pelletal bacteria cellswere collected in by centrifugation at 10000×g for 20 minutes. Thesewere suspended in 10 mM Tris-HCl pH7.7 buffer (abbreviated as buffer A,hereinafter. The pH is fully 6.1 or more, because the pH of aimedprotein is pI 6.08.) and sonicated. After storing overnight at atemperature of −80° C., the cells were sonicated again and centrifugedat 100000×g for 60 minutes. Only the most upper layer of separated threelayers was extracted.

The extract liquid was crudely purified with a 70% solution of ammoniumsulfat. The resulting extract was purified by ion exchangechromatography with anion exchange resin of beads having relatively bigparticle diameters (DEAE Sephacel of Amersham Pharmacia Biotech AB). Thebuffer A was used as equilibrium buffer, and a mixture of the buffer Aand a solution of 0.3M NaCl salt was used as eluate. Using the buffer Aand the eluate, the cells were extracted by concentration gradient. Asuitable quantity of each fraction was added dropwise on wells that HeLacells were seeded, and cell viability was evaluated in each well. Theevaluation was conducted by using WST assay using Cell Counting kits(DOJIN Laboratories). Comparing with a control, fractions showingsignificantly less viability and fractions having relatively coincidingincrease curves of the protein were evaluated along with the results ofelectrophoresis to use as sample fractions for the next purificationprocess.

Hydrophobic chromatography (Phenyl Sepharose CL-4B, Amersham PharmaciaBiotech AB) that has a different separation system from that of the nexttime was selected. Equilibrium buffer containing 1M ammonium sulfate in10 mM phosphate buffer was used. Elution buffer containing 40% ethyleneglycol in 10 mM phosphate buffer was used. After the cells wereextracted by concentration gradient, each sample was evaluated by thesame method as described in the above.

The samples extracted by the above process were extracted again by anionexchange chromatography with beads having relatively small particlediameters (RESOURCE Q of Amersham Pharmacia Biotech AB). The buffer Awas used as equilibrium buffer, and a mixture of the buffer A and asolution of 1M NaCl salt was used as elution buffer. Using thechromatography, a single band of protein of about 41000 of a molecularweight was finally obtained. The kind and order of these chromatographysmay be changed and may be further added.

After the resulting signal band was stained with Coomassie BrilliantBlue, it was transcripted on a nitrocellulose membrane or(polyvinylidene difluoride) membrane with a blotting apparatus andanalyzed with an amino acid sequencer. As the results, as described inthe above, the amino acid sequences of N ends of gene locus HP1037 of aregistered database (Helicobacter pylori 22695) matched with 95% (19bases in 20 bases). (FIG. 1)

Example 2 Amino Acid Sequences and DNA Sequences by a Gene TechnologicalProduction Method

In this example, a separation strain of Helicobacter pylori 60190 wasused. As described in the above, all gene analyses of Helicobacterpylori 22695 of different strains were already done. Homologous genelocus can be estimated by the search for the database of TIGR (TheInstitute for Genomic Research). It was found that gene locus HP1037 ofHelicobacter pylori 22695 coded the homologous protein.

From the results, the cloning of the protein of this invention wasperformed. Namely, Helicobacter pylori 60190 was used as a template,firstly, conveniently plural groups of suitable primers based on genelocus HP1037 and gene locus HP1036 of the upstream and gene locus HP1038of the downstream were formed (at 5′ site and 3′ site) to performsequening. DNA polymerase having proofreading function was used. Eachgroup of primers was constituted so as to contain sufficient mutualprimer parts, and plural sequencings from 5′ site and 3′ site wereperformed. The resulting DNA sequences were shown in SEQ ID No. 2 of thesequence listing. The amino acid sequences were shown in SEQ ID No.1.

Example 3

Expression Experiment of the Toxic Protein by Gene Recombination

E. coli was used in the expression experiment. Vector pET-30EK/LIC(producted by Novagen Co.) and E. coli BL21 (DE3) for expression wereused. The sense primer was SEQ ID No.3 inserting GACGACGACAAG at 5′ siteof the sense chain to the code of the toxic protein derived fromHelicobacter pylori 60190 that was cloned in Example 2. The antisenseprimer was SEQ ID No.4 inserting GAGGAGAAGCCCGGTTA at 5′ site. Insertinggenes were formed by a PCR method by using the above primers. Theinserted genes were prepared in the presence of 25 mM dATP and 100 mMDTT with T4 DNA polymerase to fit for LIC site of the vector and warmedin the presence of 25 mM EDTA. The formed recombinant was againsequenced to confirm that was identical with SEQ ID No. 1. It wasfurther transformed into E. coli BL21 (DE3) for expression, and culturedin a LB medium conting 30 μg/ml of kanamycin.

Shake culture was conducted to obtain about 0.4 of OD₆₀₀ at atemperature of 37° C. at 250 rpm. Isopropyl-beta-thiogalactoside wasadded to obtain the final concentration of 1 mM, and further the mixturewas shaked for 2 hours. Since the fused protein formed an inclusionbody, E. coli was collected with centrifugation, and the inclusion bodyof the protein was obtained with a BugBuster reagent and BenzonaseNuclease (both are available from Novagen Co.). The protein wasseparated by an electrophoresis method of sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE), and the corresponding single bandwas confirmed by silver stain. The protein was then refolded andcompared with the control with a WST reagent (Dojin ChemicalLaboratories Ltd. Cell Counting Kit) by using HeLa cells and the otherwarm-blooded animal cells. As the results, significant differences ofsurvival were evaluated, and it was found that the expression proteinhad equal activities to those of purified protein. It was found that theexpression protein had the same activities to the normal human gastriccells as HeLa cells of cervical cancer cells. (FIG. 2). It was alsofound that not only to the other human tissues, but also to manmaliancells the protein had broad activities. (FIG. 3, FIG. 4).

Example 4

Forming of Monoclonal Anti-M Toxic Antibody:

Expression M toxin 240 μg, which had been refolded, was subcutaneouslyadministered two times at several positions of BALB/C mouse. After 4days of final immunization, the mouse spleen was excised and pressfiltered with stainless meshes, and suspended on Eagle's modifiedminimum essential medium (MEM) to obtain a suspended solution of spleencells. As the cells used for cell fusion, Myeloma cells P3-X63. Ag 8. U1(P3U1) derived from BALB/C mouse was used as the cells used in the cellfusion.

Current topics in microbiology and immunology, 81, 1(1978

_(o). The cell fusion was conducted in accordance with the origin method

Nature, 256, 495 (1975)

. Namely, spleen cells and P3U1 were washed respectively with MEM notcontaining serum three times and mixed at 6.6:1 of a ratio of spleencells and P3U1 numbers. Cells were precipitated by centifugation at750×g for 15 minutes. All of the supernatant was removed, and theprecipitate was unfastened, 0.3 ml of 45% polyethylene glycol (PEG) 6000(Producted by Wako Junyaku Co.) was added, and the mixture was permittedto stand in a warm water tank of 37° C. for 7 minutes to perform thefusion. After the fusion, MEM in limited amounts was added to the cells,and MEM of total 15 ml was added. The mixture was centrifuged at 750×gfor 15 minutes and the supernatant was removed. The cell precipitate wassuspended in GIT medium (producted by Wako Junyaku Co.) (GIT-10% FCS)containing 10% bovine fetal serum to obtain P3U1 of 2×10⁵ per 1 ml, andseeded into 24-well multi dishes (producted by Iwaki Co.) at 1 ml perwell in 168 wells. After seeding, the cells were incubated in 5% carbondioxide incubator at a temperature of 37° C. After 24 hours, GIT-10% FCSmedium (HAT medium) containing HAT (hypoxanthin 1×10⁻⁴M, aminopterin4×10⁻⁷M and thymidine 1.6×10⁻³M) was added at 1 ml per well to initiateHAT selective cultivation. After 4 and 7 days, 1 ml of the old liquidwas discurded and the HAT selective cultivation was continued by adding1 ml of the HAT medium. Multiplication of hybridoma was found after 9days of the cell fusion, and the supernatant was collected. The antibodyvalue on the supernatant was determined by the following method. Namely,the culture supernatant 100 μl and HRP-labelled M toxin 100 μl, which isdiluted to 200-fold with buffer C, were added into each well ofmicroplates binding anti-mouse immunoglobulin antibody, and reactedovernight at a temperature of 4° C. After the plates were washed withPBS, to make microplates binding anti-mouse immunoglobulin antibody,firstly, a 0.1M carbonate buffer solution, pH9.6 containing goatanti-mouse immunoglobulin antibody (IgG fraction, producted by DAKO Co.)100 μg/ml was pipetted into 96-well microplates at 100 μl per plate, andis permitted to stand for 24 hours at a temperature of 4° C. Then, theplates were washed with phosphate-buffered saline (PBS, pH7.4), 25%Brock Ace (trademark, producted by Yukijirushi Mlk Products Co.) andPBS, pH7.2 containing 0.1% NaN₃ were pipetted at each 3001 to blockexcess binding parts of the well and treated for at least 24 hours at atemperature of 4° C. To each well of the above microplates bindinganti-mouse immunoglobulin antibody, mouse anti-serum 100 μl, which isdiluted with buffer EC [0.02M phospahte buffer, pH7.0 containing 0.2%BSA, 0.4 M NaCl, 0.4% Brock Ace, 0.05% CHAPS(3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate), 2 mM EDTAand 0.1% NaN₃], was added and reacted for 16 hours at a temperature of4° C. Then, the plates were washed with PBS, pH7.4, and HRP-labelledrefolding toxin protein 100 μl was added and reacted for 7 hours at roomtemperature. The above refolding toxin protein was prepared in the aboveExample 3 by diluting to 100-fold with buffer C [0.02M phosphate buffer,pH7.0, containing 1% BSA, 0.4 M NaCl and 2 mM EDTA]. The plates werethen washed with PBS, pH7.4, a TMB microwell peroxidase substitutesystem (KIRKEGAARD & PERRY LAB. available from Funakoshi Yakuhin) 1001μl was added, and reacted at room temperature for 10 minutes todetermine the enzyme activity on the solid phase. After the reaction wasstopped by adding 1M phosphoric add 100 μl, the absorbance at 450 nm wasdetermined with a plate reader (MTP-120, producted by Corona Co.).According these methods, the enzyme activity on the solid phase wasdetermined. As the results, 18 wells that the antibody value is foundwere selected from 123 wells, and the hybridomas were frozen and stored.Hybridomas of 6 wells, No. 4, No. 53, No. 61, No. 76, No. 101 and No.116 were cloned by dilution method. In the cloning, thymocytes of BALB/Cmouse as feeder cells were added at 5×10⁵ per well After the cloning,the antibody value of the supernatant was determined by the same method.Positive clones were No. 4, No. 101 and No. 116. These clones were asantibody-producing hybridomas of expression M toxin.

Example 5

Determination of a Class and a Subclass of Monoclonal Antibodies:

By the method described in Example 4, anti-rabbit IgG-binding microplates were formed. Namely, 0.1M carbonate buffer containing goatanti-pH9.6 was pipetted into 96-well microplates at 100 μl per well andleft at a temperature of 4° C. for 24 hours. The plates were then washedwith phosphate-buffered saline (PBS, pH7.4), 25% Block Ace (trademark,producted by Yukijirushi Milk Products Co.) and PBS, pH7.2 containing0.1% NaN₃ were pipetted at each 300 μl to block excess binding parts ofthe well and treated for at least 24 hours at a temperature of 4° C. Tothe anti-rabbit IgG antibody-binding microplates, buffer EC 50 μl andsubtype specific antibody 100 μl containing in an iso-type typing kitproducted by Bio-Rad Laboratories were added to react for one day at atemperature of 4° C. After the plates were washed with PBS, pH7.4, theculture supernatant of the hybridomas described above was added andreacted for one day at a temperature of 4° C. The plates was washed withPBS, pH7.4, and HRP-labelled refolding toxin protein 100 μl, which wasprepared in the above Example 3 by diluting to 100-fold with buffer C[0.02M phosphate buffer, pH7.0, containing 1% BSA, 0.4 M NaCl and 2 mMEDTA], was added and reacted for 6 hours at room temperature. The plateswere washed with PBS, pH7.4, and the enzyme activity on the solid phasewas determined by the method described in Example 4. As the results, thesubclasses of the monoclonal antibody porduced by these hybridomas wereNo. 4 (IgG1), No. 101 (IgG2 b) and No. 116 (IgG2a).

Example 6

A Method for Producing Mouse Ascites Fluid of Hybridoms:

Hybridomas, No. 4, No. 101 and No. 116 were mouse ascites. Mineral oil0.5 ml was parentherally administered to mice, previously. To the mice(BALB/C, female), the above s were parentherally sdministered at 1-3×10⁶cells/mouse, and the ascites containing the antibody were collectedafter 6-20 days. The monoclonal antibody was purified from the obtainedascites with a protein-A column. Namely, the ascites about 25 ml werediluted with the same volume of binding buffer (3.5M NaCl, 1.5M glycincontaining 0.05% NaN₃, pH9.0), the solution was precipitate to arecombinant protein-A-agalose (Repligen Co.) column that was previouslyequilibrated with binding buffer, the specific antibody was eluted withelution buffer (0.1M citric acid buffer, pH3.0 containing 0.05% NaN).The elution liquid was dialyzed with PBS, pH7.4 at a temperature of 4°C. for 2 days, and filtered for removing bacilli with a filter of 0.22μm (producted by Millipore Co.) or stored at a temperature of −80° C.

Example 7 Preparation of Polyclonal Antibody M Toxin

Cytotoxic protein M toxin of this invention 4.1 mg was mixed withFreund's complete adjuvant and the mixture was subcutaneously immunizedto a rabbit. After one week, the same amount of the M toxin was mixedwith Freund's incomplete adjuvant and the mixture was furthersubcutaneously immunized to the rabbit. After the immunization,collected blood was centrifuged to remove hemocyte components, andanti-serum was obtained.

Example 8

Analysis of M toxin by Western Blotting:

SDS-Sample Buffer containing 2-mercaptoethanol was added to thesupernatant obtained in Example 3, the mixture was electrophoresed withPeptide-PAGE (TEFCO) and electrically transferred to a PVDFmemmbrane(Amersham). Each antibody (2 μg/ml) obtained in Example 4 and Example 6was used as primary antibody. HRP (Horseradishperoxidase)-labelled-anti-rabbit and anti-mouse IgG antibodies(2000-fold dilution; Dako) was used as secondary antibody. Thecoloration was performed with an ECL Western Blot Detection System(Amersham). As the results, it was confirmed that each primary antibodyrecognized the expression protein. (FIG. 5).

Example 9

Active carbon (0.2˜0.1 mm of diameter), CM cellulose and calciumalginate, each 0.5 ml, were charged in three colums. After equilibratingwith Tris buffer 10 mM, pH7.7, 0.5 ml of M toxin 400 nM having refoldedactivity was added into each column, and these column was plugged andallowed to stand for 60 minutes at a temperature of 25° C. Meanwhile,Tris buffer alone and Tris buffer containing the same M toxin wereallowed to stand under the same conditions. All columns were thenopened, each 100,u 1 dropping from columns was separated, further, eachTris-buffer 0.5 ml was added to each column, and each 100 μl wascollected. Whole blood of normal adults was centrifuged 2-3 times at800×g for 10 minutes until the supernatant became transparent. Theprecipitated erythrocytes 10 μl added to 10 mM Tris buffer 990 μl wasused as a positive control. Similarly, the precipitated erythrocytes 10μl added to 0.9% salt 10 mM Tris buffer was used as a negative control.Samples were collected from the above columns. Erythrocytes of 1 volumeper 100 volume of the sample were added to the samples. The controls andthe samples were compared by the eluted hemoglobin relativeconcentration with a multiple plate reader (Biorad) by measuring as anabsorbance of 415 nm. (FIG. 6).

Example 10

The ratios that HeLa cells (human cancer cells of the uterine cervix)were exposed to the fractions from each columns of Example 9 and theviability were determined by a WST method using a tetrazolium salt. HeLacells were cultured in 96-well plates for 24 hours at a ratio of 10000cells per well. A negative control of culture liquid only, each sampleliquid, and a positive control of 20 nM of the toxin protein wererespectively added to each well, and thsese were cultured for 12 hoursatat a temperature of 37° C. Using a Cell Counting Kit (DOJIN LABORATORIESLtd. Co.), the ratios of cell viability detected by a WST method weremeasured as an absorbance of 415 nm wavelength. (FIG. 7, FIG. 8).

INDUSTRIAL APPLICABILITY

The protein and the partial peptide of this invention and the like canbe used, for example, as treating agents of cancers. The antibody ofthis invention can be used to identify the protein of this invention inthe blood, tissues, urine and excrements collected from test patientsand to confirm the Helicobacter pylori infection. It is further used forquantification of the protein of this invention. The protein of thisinvention can be used as a screening agent of the compounds increasingor inhibiting the activities of the protein of this invention.

1. An isolated cytotoxic protein comprising a protein having the aminoacid sequence represented by SEQ ID NO:1.
 2. The isolated cytotoxicprotein of claim 1, wherein the protein is produced with Helicobacterpyloici.
 3. The isolated cytotoxic protein of claim 1, wherein theprotein is obtained by culturing a transformant which is transformedwith a recombination vector containing DNA of SEQ ID NO:2 coding saidcytotoxic protein.
 4. The isolated cytotoxic protein of claim 3, whereinthe transformant is deposited by deposition No. FERM BP-8218 at theInternational Patent Organism Depository, National Institute of AdvancedIndustrial Science and Technology.
 5. A composition comprising saidisolated cytotoxic protein of claim 1 and a pharmaceutically acceptablecarrier.
 6. A kit for screening a compound or a salt thereof thatinhibits the activity of the isolated cytotoxic protein of claim 1,comprising said isolated cytotoxic protein.